Sequence-specific hybridization of oligonucleotide probes has long been used as a means for detecting and identifying selected nucleotide sequences, and labeling of such probes with fluorescent labels has provided a relatively sensitive, nonradioactive means for facilitating detection of probe hybridization. Recently developed detection methods employ the process of fluorescence energy transfer (FET) for detection of probe hybridization rather than direct detection of fluorescence intensity. Fluorescence energy transfer occurs between a donor fluorophore and an acceptor dye (which may or may not be a fluorophore) when the absorption spectrum of one (the acceptor) overlaps the emission spectrum of the other (the donor) and the two dyes are in close proximity. The excited-state energy of the donor fluorophore is transferred by a resonance dipole-induced dipole interaction to the neighboring acceptor. This results in quenching of donor fluorescence. In some cases, if the acceptor is also a fluorophore, the intensity of its fluorescence may be enhanced. The efficiency of energy transfer is highly dependent on the distance between the donor and acceptor, and equations predicting these relationships have been developed by Forster (1948. Ann. Phys. 2:55-75). The distance between donor and acceptor dyes at which energy transfer efficiency is 50% is referred to as the Forster distance (R.sub.O). Other fluorescence properties may also be dependent on the proximity of a donor and an acceptor, e.g., fluorescence lifetime of the donor and/or acceptor, fluorescence polarization and fluorescence anisotropy.
Energy transfer and other mechanisms which rely on the interaction of two dyes in close proximity to produce a change in a fluorescence property are an attractive means for detecting or identifying nucleotide sequences, as such assays may be conducted in homogeneous formats. Homogeneous assay formats are simpler than conventional probe hybridization assays which rely on detection of the fluorescence of a single fluorophore label, as heterogenous assays generally require additional steps to separate hybridized label from free label. Typically, FET and related methods have relied upon monitoring a change in the fluorecence properties of one or both dye labels when they are brought together by the hybridization of two complementary oligonucleotides. In this format, the change in fluorescence properties may be measured as a change in the amount of energy transfer or as a change in the amount of fluorescence quenching. In this way, the nucleotide sequence of interest may be detected without separation of unhybridized and hybridized oligonucleotides. The hybridization may occur between two separate complementary oligonucleotides, one of which is labeled with the donor fluorophore and one of which is labeled with the acceptor. In double-stranded form there is decreased donor fluorescence (increased quenching) and/or increased energy transfer as compared to the single-stranded oligonucleotides. Several formats for FET hybridization assays are reviewed in Nonisotopic DNA Probe Techniques (1992. Academic Press, Inc., pgs. 311-352). Alternatively, the donor and acceptor may be linked to a single oligonucleotide such that there is a detectable difference in the fluorescence properties of one or both when the oligonucleotide is unhybridized vs. when it is hybridized to its complementary sequence. In this format, donor fluorescence is typically increased and energy transfer and quenching are decreased when the oligonucleotide is hybridized. For example, a self-complementary oligonucleotide labeled at each end forms a hairpin which brings the two fluorophores (i.e., the 5' and 3' ends) into close proximity where energy transfer and quenching can occur. Hybridization of the self-complementary oligonucleotide to its complement on a second oligonucleotide disrupts the hairpin and increases the distance between the two dyes, thus reducing quenching. A disadvantage of the hairpin structure is that it is very stable and conversion to the unquenched, hybridized form is often slow and only moderately favored, resulting in generally poor performance. A "double hairpin" scheme is described by B. Bagwell, et al. (1994. Nucl. Acids Res. 22:2424-2425). Kramer and Tyagi (1996. Nature 14:303-308) describe a hairpin with the detector sequence in a loop between the arms of the hairpin.
Prior art methods may lack efficiency in the energy transfer itself, and it has often been difficult to achieve adequate spectral resolution to detect meaningful changes in fluorescence. In many methods which monitor fluorescence quenching a small amount of hybridization produces only a small decrease in fluorescence which must be detected in the presence of high levels of background. These methods also suffer from lack of detection sensitivity.
Aptamers are DNA or RNA molecules which bind specific molecular targets. Large populations of randomly generated oligonucleotides may be enriched in aptamers by known in vitro selection and amplification processes. Of particular interest is a single-stranded DNA aptamer which binds thrombin (L. C. Bock, et al. 1992. Nature 355:564-566). These thrombin binding aptamers were found to contain the conserved consensus sequence GGNTGGN.sub.2-5 GGNTGG (SEQ ID NO: 1) and inhibited thrombin-catalyzed fibrin-clot formation. Analysis of the structure of this molecule has revealed a symmetrical structure containing two tetrads of guanosine base pairs connected by three loops (1993. K. Y. Wang, et al. Biochemistry 32:1899-1904; 1993. R. F. Macaya, et al. PNAS 90:3745-3749; 1994. P. Schultze, et al. J. Mol. Biol. 235:1532-1547; 1996. J. A. Kelly, et al. J. Mol. Biol. 256:417-422). This characteristic structure is commonly referred to as a "G-quartet," "G-quadruplex" or "G-tetraplex" structures. E. Dias, et al. (1994. J. Am. Chem. Soc. 116:4479-4480) report a similar sequence in which the G-quartet structure is maintained when the length of the oligonucleotide between the G pairs is increased.
A fluorophore is a dye or chemical moiety which can be made to fluoresce. This includes dyes which fluoresce in response to chemical treatment, excitation by light or in biological systems.
A donor or donor fluorophore is a fluorophore which has a fluorescence emission spectrum which overlaps the absorption spectrum of the second dye or chemical moiety.
An acceptor or acceptor dye is a dye or other chemical moiety which absorbs light emitted by a donor fluorophore.